Treatment of cancer using lipoic acid in combination with ascorbic acid

ABSTRACT

Lipoic acid and/or its water soluble salt is used to treat cancer, alone or in combination with ascorbic acid (vitamin C). Alone or in combination, it was shown to be effective on in vitro tumors and mouse tumors. The agents can be administered safely, and have been used effectively in case studies.

This is a Divisional of U.S. application Ser. No. 09/249,872, filed Feb.16, 1999.

FIELD OF THE INVENTION

This invention relates generally to methods of cancer therapy andparticularly the use lipoic acid as a therapeutic agent administered incombination with ascorbic acid. Ascorbate has been shown to beselectively toxic toward tumor cells, but at doses that are too high tobe achieved clinically. Both lipoic acid and its water-soluble sodiumsalt enhance the efficacy of sodium ascorbate against three-dimensionalin vitro tumors and in mouse tumors. These agents can be administeredsafely to patients, and in preliminary trials have been shown tostabilize or resolve disease.

BACKGROUND OF THE INVENTION

The most common methods currently in use for the treatment of cancerinclude surgery, radiation therapy, and chemotherapy. While thesetherapies are successful for some forms of the disease, they are farfrom universally successful in curing cancer. Moreover, traditionaltherapeutic regimens often cause adverse side effects such as nausea,vomiting, cardiac toxicity, bone marrow suppression, and secondarycancer. Vitamin C (ascorbic acid, ascorbate) has been proposed as analternative to chemotherapy or as an adjuvant to lessen side effectsassociated with it. (For the purposes of this application, a referenceto ascorbic acid includes the anionic component, ascorbate whether as anacid or one of the pharmaceutically acceptable salt thereof, mostnotably including sodium ascorbate and calcium ascorbate, any of whichare included in a reference to “ascorbic acid” or “ascorbate”). Ascorbicacid has been thought by some to improve immune response and to preventtumor spreading by strengthening extracellular matrix, but thesetheories have not as yet been conclusively proven. Clinical trials withascorbate at doses on the order of 10 g/day were successful in somecases, but not in others. At very high doses, ascorbic acid ispreferentially toxic to tumor cells. This preferential toxicity isunderstood to relate to the ascorbate mediated production of hydrogenperoxide, which is more toxic to tumor cells due to the lower levels ofcatalase typically present in tumor cells as compared to normal cells.High dose intravenous ascorbate has thus been suggested for thetreatment of cancer, as described in U.S. Pat. No. 5,639,787.

Critical to the use of high dose intravenous ascorbate as an anti-canceragent is the ability to clinically achieve plasma ascorbate levelssufficient to kill tumor cells. Previous measurements of ascorbateplasma levels following intravenous infusion demonstrate concentrationsgreater than those needed to kill tumor cells grown in monolayercultures in vitro. However, much higher levels of ascorbate are requiredto kill tumor cells grown as three-dimensional in vitro tumors. Usingthe hollow fiber “solid” tumor model, it has been observed thatascorbate concentrations in excess of 500 mg/dL may be required toeffectively treat tumors. Currently, the maximum plasma ascorbic acidconcentration generally achievable in humans by intravenous infusion isroughly 500 mg/dL, a peak level that drops off sharply over a relativelyshort period of time. Although ascorbate is relatively innocuous tohuman patients, the need for higher plasma concentrations demonstrates aneed either to safely raise effective plasma levels of ascorbate or toincrease the cytotoxic effectiveness of ascorbate toward cancer cells todecrease the required dosage.

SUMMARY OF THE INVENTION

One object of the invention is to provide a method for treating cancerby administering lipoic acid and/or a water soluble salt of lipoic acid.Preferably the lipoic acid is administered at 100-1000 mg/day. Morepreferably at 300-600 mg/day.

A further object of the invention is to provide a method for treatingcancer wherein the lipoic acid or combined therapy is used incombination with another therapy. Preferably the lipoic acid is used incombination with ascorbic acid (vitamin C). Preferably the ascorbic acidis administered intravenously at 15-700 g/week, more preferably 50-200g/week. The preferred ratio of ascorbic acid to lipoic acid is fromabout 1:1 to about 3500:1, more preferably from about 10:1 to about100:1.

A further object of the invention is to provide a pharmaceuticalcomposition for treating cancer in a human or other animal comprisinglipoic acid and ascorbic acid in an effective dose. The preferred ratioof ascorbic acid to lipoic acid is from about 1:1 to about 3500:1, morepreferably from about 10:1 to about 100:1. Preferably the dose ofascorbate is 2-250 g per infusion per day. Preferably the concentrationof lipoic acid is 100-1000 mg.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention comprises the treatment of cancer byadministering ascorbic acid in combination with lipoic acid insufficient amounts to achieve a level of concentration in the patient'splasma that is cytotoxic to the cancer cells as demonstratedexperimentally using in vitro culture models that mimic the in vivosolid tumor. The basis of the present invention is the discovery,through experimentation, that the concentration of ascorbate required toinduce cytotoxicity in an in vitro solid tumor model is significantlyreduced when the ascorbate is administered in combination with lipoicacid. Specifically, a ten to one ratio of ascorbate to lipoic acid wasfound to reduce the ascorbate concentration required to kill fiftypercent of the tumor cells treated by roughly a factor of five over thatnecessary to achieve the same cell killing with ascorbate alone, i.e. asynergistic effect is achieved. This was a surprising result, sincelipoic acid is a free radical scavenger that has been shown to inhibitascorbate mediated hydrogen peroxide generation by erythrocytes. Theimportance of using lipoic acid in combination with ascorbate is thatthe level of ascorbate required for effective tumor toxicity is reducedto a level that has been successfully replicated in vivo, i.e. in theplasma of patients to whom the combination is administered throughintravenous infusion.

Target levels of ascorbic acid and lipoic acid can be set according tothe type of cancer afflicting the patient using in vitro studies ofcytotoxicity in similar cell lines. More accurate target levels can beachieved by in vitro experimentation of malignant cells taken from thepatient if such are available. This invention is not specific to a givenascorbate dose or schedule of intravenous administration. The use of oneto eight hour infusions or continuous infusions with the aid of infusionpumps may be indicated. Nor is this invention specific to the vehicle oflipoic acid administration.

Further features and advantages of the present invention will becomeapparent to those of skill in the art in view of the detaileddescription of the preferred embodiments which follows when consideredtogether with the attached drawings and claims.

DL-α-Lipoic Acid (DL-6,8-Thioctic Acid) is a lipophilic antioxidant thatcan be readily obtained commercially in clinical or research reagentgrade. While the lipophilic form is favorable for in vivo use, some invitro experiments were carried out using a water soluble salt producedfrom lipoic acid by mixing the acid with sodium bicarbonate in aqueoussolution and then drying by lyophilization. For the purposes of thisapplication, a reference to lipoic acid includes both the lipophilicacid and the water soluble salts.

Although other material and method similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.Example 1 describes toxicity studies that were done on cell monolayersto determine the feasibility of using the combination of ascorbic acidand lipoic acid to treat cancer.

EXAMPLE 1 Toxicity Tests in Cell Monolayers

The toxicity of ascorbic acid and lipoic acid (sodium salt) was testedin immortalized cell monolayers of both tumor and normal tissue origin.In solid tumors, proliferating cells are sometimes more sensitive totreatment than quiescent, non-proliferating cells. To investigate thisin cell monolayers, we used sparse plating densities (6K/well in a 96well plate) to allow cell proliferation and confluent densities(24K/well in a 96 well plate) to simulate quiescent cells. Cells wereplated in 96 well plates at sparse or confluent densities and thenincubated four days in growth medium supplemented with various doses ofantioxidants. After incubation, cell numbers were determined by standardcalorimetric techniques. Data were analyzed by scaling the cell numberat any given antioxidant dose to that of the control and fitting thedose-response data to a sigmoid curve. Values of LC₅₀, the dose requiredto reduce cell number by fifty percent relative to untreated controls,were computed from the sigmoid data fit. Results are given in theaccompanying table.

Human Cell Types and Densities Used LC₅₀ (mg/dL) Line Origin DensityAscorbate Lipoic Acid ECV- Endothelial Sparse 149 ± 16 127 ± 15 304Confluent no data no data SW620 Colon Adenocarcinoma Sparse 30 ± 4 34 ±2 Confluent  39 ± 14 24 ± 2 SK- Melanoma Sparse 15 ± 2  70 ± 20 MELConfluent  78 ± 16 22 ± 2 Mia Pancreatic Carcinoma Sparse 53 ± 6  80 ±21 PaCa Confluent 308 ± 33 109 ± 45 MCF-7 Breast Carcinoma Sparse 12 ± 1125 ± 31 Confluent 23 ± 3  60 ± 245

These data indicate that lipoic acid is toxic to cells at high doses;moreover, the LC₅₀ values for the four tumor cell lines studied (SW620,SK-MEL, Mia PaCa, and MCF-7) tended to be lower than those for the cellline derived from normal tissue (ECV-304), suggesting a greater toxicitytoward tumor cells than normal cells. This was a surprising result,since there are no reports of hydrogen peroxide generation by lipoicacid; in fact, lipoic acid has been shown to inhibit ascorbate mediatedhydrogen peroxide generation by erythrocytes. There seems to be littledependence of lipoic acid LC₅₀ on cell density, suggesting that itstoxic effect is not dependent upon cell proliferation. In contrast, thedata for ascorbate suggest that confluent cells were more resistant.

To determine whether the combination would work on solid tumors, invitro tests were performed in Example 2.

EXAMPLE 2 Combination Therapy Tests in Hollow Fiber Solid in vitroTumors

Experiments combining ascorbate and lipoic acid were carried out usingthe SW620 hollow fiber solid tumor model. In this in vitro tumor model,SW620 cells grow in three dimensions, forming a cylindrical tumor massroughly 500 μm in diameter that is similar in terms of microenvironmentand proliferative heterogeneity to a micro-region of a solid in vivotumor. As with solid tumors, drugs must penetrate the inner regions ofhollow fiber tumors by diffusion. This model thus provides a moreformidable test for drug efficacy than cell monolayers.

SW620 hollow fiber solid tumors were grown as previously described.Briefly, PVDF hollow fibers (500 KD molecular mass cutoff, 500 mm insidediameter with a roughly 100 mm wall thickness, Spectrum Medical Co.)were prepared for cell culture use by soaking them for one week inmethanol and then storing them in cell culture medium (RPMI mediumsupplemented with antibiotics, glutamine, and 10% fetal calf serum).SW620 colon carcinoma cells were prepared in cell culture medium at aconcentration of 10⁷ cells/ml and injected into the hollow fibers. Thefibers were then heat sealed at roughly 2 cm intervals to trap the cellsinside them. SW620 hollow fibers were then cultured in Petri dishes forthree days. After three days, the hollow fibers are transferred tostirred medium either by moving them to spinner flasks containing 50 mlcell culture medium stirred at 150 rpm by a magnetic stirrer or bytransferring them to six-well plates placed on an orbital shaker at 150rpm. Cell culture medium was replenished on days three and six. On dayeight or nine, SW620 hollow fiber tumors were transferred totwenty-four-well plates containing cell culture medium supplemented withvarious doses of ascorbate and lipoic acid or lipoic acid sodium. Afterone or two days incubation on an orbital shaker, the hollow fiber tumorswere rinsed and the cells were extruded out of the fibers with trypsin.The cylindrical tumor cell mass was exposed to trypsin while pipettingup and down to form a single cell suspension. Cell culture medium wasthen added to stop trypsin action. Surviving fractions were determinedas previously described. Briefly, cells were transferred toninety-six-well plates at a concentration of 5000 cells per well andincubated for six days. The surviving cell population was then measuredusing the SRB colorimetric assay. Surviving fraction was expressed asthe ratio of the SRB staining at a given dose to that for cells fromuntreated hollow fiber tumors.

The effect of lipoic acid on SW620 hollow fibers was analyzed usingdose-response curves for SW620 colon carcinoma tumor cells grown fornine days as hollow fiber tumors and treated for 48 hours with sodiumascorbate alone or in combination with lipoic acid. Lipoic acidconcentrations of 5 mg/dl or 20 mg/dl were tested. The survivingfractions without ascorbate (denoted SF_(O)) were analyzed along withthe doses of ascorbate required to reduce the surviving fraction to halfthe SF_(O) value, denoted LC₅₀. Standard deviations for SF_(O) and LC₅₀values were analyzed by the curve-fitting program KalediaGraph (SynergySoftware). In this experiment the lipoic acid concentration was keptconstant while the ascorbate dose was varied. It was clear from thesedata that lipoic acid itself, at the two concentrations tested,exhibited a toxic effect against tumor cells grown in three dimensions;moreover, lipoic acid amplified the toxic effects of ascorbate, causingreductions in surviving fraction at much lower doses than for ascorbatealone. The LC₅₀ value for ascorbate in the presence of 20 mg/dL lipoicacid was roughly half that in the absence of ascorbate, and thedifference was statistically significant. This effect on ascorbateefficacy was not expected a priori. The ability of lipoic acid toscavenge free radicals that would otherwise be converted by ascorbate tohydrogen peroxide might suggest a protective effect rather than a toxicone. Lipoic acid, like other lipophilic antioxidants such as vitamin E,may recycle ascorbate from the reduced dehydroascorbate form. However,the effect of this recycling is uncertain: while recycled ascorbate mayincrease cytotoxicity due to increased hydrogen peroxide generation, anycytotoxic effects generating from the actions of ascorbate oxidationproducts will be reduced. Thus, the mechanism of lipoic acid'senhancement of ascorbate toxicity is unknown, though it may aid inkilling quiescent cells that are resistant to ascorbate, as suggested bythe cell monolayer data above.

Additional tests were carried out by varying the lipoic acidconcentration with the ascorbate concentration while using a 100:1 ratioof ascorbate to lipoic acid (to yield a lipoic concentration from 5 to10 mg/dL at the LC₅₀). The effect of lipoic acid in series ofexperiments was to again decrease the ascorbate LC₅₀ against SW620hollow fiber tumors by roughly a factor of two. Since in vitroexperiments with lipoic acid were limited by its hydrophobic properties,we used the hydrophilic sodium salt to lipoic acid to provide higherascorbate to lipoic acid ratios. Specifically, a 10:1 ratio of ascorbateto the sodium salt of lipoic acid was tested against SW620 hollow fibertumor cells. Dose response curves for SW620 colon carcinoma tumor cellsgrown for 9 days as hollow fiber tumors and treated for 48 hours withsodium ascorbate alone or in combination with the sodium salt of lipoicacid were prepared. In the combination treatment, the lipoic acid toascorbate ratio was 1:10. Data from three replicate experiments werepooled. The doses of ascorbate required to reduce the surviving fractionto half the control value were denoted as LC₅₀. Standard deviations onLC₅₀ values were estimated by the curve-fitting program kaleidaGraph(Synergy Software). The results, suggest that lipoic acid sodium at thisratio can reduce the ascorbate LC₅₀ by roughly a factor of five, from490 mg/dL in the absence of lipoic acid to 90 mg/dL in the presence oflipoic acid. The importance of this result is indicated by comparing theconcentrations of ascorbate required for cytotoxicity in hollow fibertumor to the doses that can be obtained in blood plasma duringintravenous infusion. A plasma ascorbate concentration of 490 mg/dL ismuch more difficult to achieve and sustain than a value of 90 mg/dL.

To determine the feasibility of in vivo administration of thecombination therapy, in Example 3 serum levels were determined afterintravenous administration.

EXAMPLE 3 Studies of Ascorbate Pharmacokinetics: Implications for thePresent Invention

Pharmacokinetic measurements and compartmental analysis were used todetermine the potential relevance of lipoic acid and ascorbatecombination therapy in the clinic. A seventy-five year old Caucasianmale with diagnosed metastatic prostate cancer was infused intravenouslywith 65 grams sodium ascorbate in sterile water over an eighty minuteperiod. Blood samples were taken at regular intervals over a twenty-fourhour period, frozen, and then analyzed for ascorbate content using acolorimetric assay. The ascorbate plasma concentration was analyzed in aprostate cancer patient given a 60 gram intravenous infusion of sodiumascorbate over an 80 minute period. Plasma ascorbate levels measuredover a 24 hour period were fit to a two compartment, four parameterpharmacokinetic model of ascorbate uptake and clearance from plasma andtissue The following parameter values were obtained: K_(X)=0.124 min⁻¹,K₁=0.124 min⁻¹, K₂=0.038 min⁻¹. The plasma volume V_(p), was fixed at 30dl. A peak plasma value of 460 mg/dL was obtained, but it quicklydiminished with time. Since levels might be sustained longer withcontinuous infusions, the two-compartment model was used, andhypothetical plasma ascorbate curves were computed for the case ofcontinuous twenty-four hour infusions of various ascorbate doses.Predicted plasma ascorbate levels from a two compartment, four parameterpharmacokinetic model of ascorbate transport and clearance in blood.Plasma vs. time curves are given for five different infusion doses: 25,50, 75, 100, and 125 grams per day. The infusion rate, G(t), is set tovarious values as shown in the figure. Infusion is modeled ascontinuous. Parameter values for the compartmental model are as follows:K_(X)=0.124 min⁻¹, K₁=0.124 min⁻¹, K₂=0.038 min⁻¹. The plasma volumeV_(p), was fixed at 30 dl. A phase one clinical trial is currently underway to assess the safety of continuous ascorbate infusions, and iscurrently near completion. Doses up to 50 g/day were tested and foundsafe. From the modeling results in EXAMPLE 3, this dose is expected toproduce plasma concentrations of roughly 52 mg/dL after twenty-fourhours. Based on the data in EXAMPLE 2, SW620 hollow fiber tumor cellsexposed to 52 mg/dL ascorbate for forty-eight hours would have asurviving fraction of greater than ninety percent; in contrast, the sametumor cells exposed to the same ascorbate dose combined with lipoic acidwould have a surviving fraction of only sixty percent.

Thus, dose-response data with hollow fiber in vitro tumors, combinedwith pharmacokinetic data and modeling, suggest that ascorbate is toxicat clinically achievable doses when lipoic acid is used in combinationwith it. Therefore, tests were performed in an in vivo cancer model.

EXAMPLE 4 Toxicity Tests in a Murine Tumor Model

Lipoic acid alone or in combination with ascorbate (a 10:1 ascorbate tolipoic acid ratio) was tested against mouse tumors at the Beijinginstitute. C57 mice were divided into groups of twenty and inoculated byB16 melanoma cells subcutaneously. After forty-eight hours, treatmentswere administered once every other day for a five week period bysubcutaneous injection near the tumor site. Tumor size was measured ondays ten, seventeen, and twenty-four of treatment. The tumors in micetreated with lipoic alone or in combination with ascorbate weresignificantly smaller than those in the control group. Data at daytwenty-four are given in the accompanying table:

Treatment Group Relative Tumor Size Animal Survival Control 100% 14/20(70%) Lipoic Acid Alone 60% (p < 0.01) 18/20 (90%) Lipoic Acid andAscorbate 51% (p < 0.01) 13/20 (65%)

Lipoic acid was toxic to mouse tumors with or without ascorbate,although animal survival rates were better for the combination. Whilethe results for lipoic acid alone are surprising, the experiment overallconfirms the utility of using lipoic acid as an anti-cancer agent incombination with ascorbate. Therefore, in the following case studies,combination therapy was initiated.

EXAMPLE 5 Case studies using the combination therapy

Case Study #1:

A sixty-five year old Caucasian female began treatment at our clinic onDecember 1^(st), 1998. In the previous year she was diagnosed by heroncologist as having low-grade small-cell malignant lymphoma with theinvolvement of the bone marrow. The circulating platelet count is a goodindicator of metastatic burden in the bone marrow, as the expectedcourse for this disease is for an unremitting decline in circulatingplatelet levels. Indeed, the patient indicated in her correspondenceswith our clinic that her platelet counts had fallen from 239,000 to160,000 in the six months immediately prior to the commencement oflipoic acid therapy. She was placed on a regimen of 300 mg Lipoic Aciddaily along with other nutritional supplements to accommodate hernutritional deficiencies. Two months later, She was started onintravenous vitamin C therapy, at 50 grams twice per week. Six monthsafter her first visit, her ascorbate dose was increased to 75 gramstwice per week. The patient's platelet levels remained relatively stableduring the course of treatment: her counts never went below 100,000 asnormally expected with this type of cancer. Six months after initiatinglipoic acid treatments, her platelet count was stable at 190,000.

Case Study #2:

A 70-year-old Caucasian male was diagnosed with pancreatic cancer onDecember 3^(rd), 1996. He opted to undergo traditional chemotherapy inJanuary, 1997. One tumor marker for pancreatic cancer is a carbohydrateantigen known as CA-19-9. The patient's antigen level decreasedinitially after the first chemotherapy treatment; however, by October,1997, the his CA-19-9 level had increased to a value of 7,400 units/mLserum. At that time he was placed on a regimen of 15 grams intravenousvitamin C, given two times weekly and 300 mg oral lipoic acid, taken twotimes per day, as well as other nutrient supplements that the patientwas deficient in. One month later his dose of Vitamin C increased to 25grams two times per week. Within two months the patient's antigen levelhad dropped to 3,200 units/mL serum. At that time his dosage of VitaminC was increased incrementally 30 grams, 50 grams, and finally 75 grams.In January, 1998, the patient opted to stop chemotherapy whilecontinuing lipoic acid and ascorbate treatments. The levels of CA-19-9however continued to fall. In March, 1998, the patient's CA-19-9 was 700units/ml serum. The patients CA-19-9 levels during the time course oftreatment with ascorbate and lipoic acid decreased. The levels ofCA-19-9, a pancreatic cancer marker, expressed as units per mL of serum,were analyzed with time. Lipoic acid was given orally at a dose of 300mg administered twice daily. Ascorbate was give intravenously at aninitial dose of 15 grams per week. This dose was incrementally increasedto 25 grams twice per week, one month after the onset of therapy, andthereafter was incrementally increased to 30, 50, and finally, 75 gramstwice per week. The patient discontinued ascorbate and lipoic acidtherapy in March, 1998, after six months of treatment. He died fourmonths later.

Case Study #3:

A 53-year-old Caucasian male was diagnosed with colorectal cancer withliver metastases, and underwent surgery to remove the largest of theliver metastases. The patient was started on low dose 5-fluorouracilchemotherapy, intravenous vitamin C, and oral lipoic acid. He was alsogiven various nutritional supplements to combat diagnosed deficiencies.The vitamin C dose was gradually increased to 100 grams, administeredtwice weekly by intravenous infusion. He continued therapy until March,1998, when he was declared to be cancer-free. On his last follow-up inDecember, 1998, the patient was still cancer-free.

What is claimed is:
 1. A method for the treatment of cancer in a mammalhaving cancer consisting essentially of the steps of: administering asynergistically effective concentration of lipoic acid or its salt incombination with ascorbic acid (Vitamin C) or its salt, wherein saidcancer is sensitive to said combination, and wherein the combination isin a ratio of lipoic acid to ascorbic acid of about 1:10 to about1:3500, and wherein the action of the lipoic acid potentiates the actionof the ascorbic acid.
 2. The method of claim 1, wherein said effectiveamount of ascorbic acid is 15 g/week to 700 g/week.
 3. The method ofclaim 2, wherein said effective amount of ascorbic acid is 15 g/week to200 g/week.
 4. The method of claim 3, wherein said effective amount ofascorbic acid is 50 g/week to 500 g/week.
 5. The method of claim 4,wherein the ratio by weight of lipoic acid to ascorbic acid is fromabout 1:100 to about 1:2500.
 6. The method of claim 1, wherein saidmammal is a human.
 7. The method of claim 1, wherein the ratio of lipoicacid to ascorbic acid is 1:50-1:3500.
 8. The method of claim 1, whereinthe ratio of lipoic acid to ascorbic acid is 1:100-1:3500.
 9. Apharmaceutical composition for treating cancer in a mammal havingcancer, said composition comprising: a synergistically effectiveconcentration of ascorbic acid (vitamin C) or its salt in combinationwith lipoic acid or its salt in a ratio of lipoic acid to ascorbic acidof 1:10 to about 1:3500; and a pharmaceutically acceptable vehicle,wherein said cancer is sensitive to said pharmaceutical composition, andwherein the action of the lipoic acid potentiates the action of theascorbic acid.
 10. The pharmaceutical composition of claim 9, whereinthe ratio by weight of lipoic acid to ascorbic acid is from about 1:2500to about 1:3500.
 11. The pharmaceutical composition of claim 9, whereinthe amount of ascorbic acid is 2-250 g.
 12. The pharmaceuticalcomposition of claim 9, wherein the amount of ascorbic acid is100-25,000 mg.
 13. The composition of claim 9, wherein the ratio oflipoic acid to ascorbic acid is 1:50-1:3500.
 14. The composition ofclaim 9, wherein the ratio of lipoic acid to ascorbic acid is1:100-1:3500.